Cytoskeletal Components of the Cell
The cytoskeleton plays an important role in the growth, division, and migration of eukaryotic cells. Changes in cellular morphology, the repositioning of internal organelles, and cellular migration all depend on complex networks of protein filaments that traverse the cytoplasm.
These protein filaments fall into three main categories according to their size: microtubules, intermediate filaments, and microfilaments. Both microtubules and microfilaments are made of globular subunits which can quickly polymerize and depolymerize in the cell resulting in movement and morphological changes. Intermediate filaments are made of fibrous protein subunits and tend to be more stable with longer half-lives than most microtubules and microfilaments.
Current theory holds that cells have a pool of unpolymerized globular subunits in the cytoplasm which is used to rapidly form the cytoskeletal microtubules and microfilaments. Microtubules are formed by a dimer of tubulin proteins which take on a helical shape to form filaments. Similarly, microfilaments comprise actin proteins which agglutinate together to form elongated filaments. In addition to these fibers, the cytoskeleton is also made up of many other components for linking the filaments to each other or to the plasma membrane. Many cytoplasmic components can influence the rate of filament polymerization or depolymerization. Also, drugs have been discovered which affect the rate of filament polymerization and lead to either abnormal accumulations of protein filaments or unpolymerized globular subunits.
Taxol, colchicine, vinblastine, cytochalasin-B, and cytochalasin-D are all well known disruptors of cytoskeletal development. Taxol inhibits depolymerization of the microtubule filaments, while vinblastine and colchicine inhibit microtubule polymerization. Griseofulvin is another drug that interferes with microtubule function, although the mechanism of this interference is not yet established. Cytochalasin-B and cytochalasin-D are inhibitors of microfilament networks.
Diseases Involving Cyst Formation
There are many human diseases which result in the formation of cysts which contain either semi-solid or fluid material. The contents of a cyst sometimes derive from normally retained fluid (e.g. a sebaceous cyst can contain fluid from a blocked sebaceous gland) or from a parasitic infection. Benign cysts can occur in the ovary, spleen, lungs, kidney and liver, where they are often congenital. Some congenital cysts result from fetal malformations and developmental failure while others are direct results of a disease state.
The polycystic kidney diseases (PKD) are a group of disorders characterized by a large number of cysts distributed throughout dramatically enlarged kidneys. The resultant cyst development leads to impairment of kidney function and can eventually cause kidney failure. In humans, PKD can be inherited in autosomal dominant (ADPKD) or autosomal recessive (ARPKD) forms.
ADPKD is the most common dominantly inherited kidney disease of humans, while ARPKD occurs relatively rarely. Clinically, ADPKD represents a major cause of chronic renal failure in humans and accounts for 10% of all patients requiring chronic dialysis or renal transplantation. Currently, 500,000 Americans and 5 million people worldwide are estimated to be afflicted with PKD. In the U.S. this represents an annual health care cost of nearly one billion dollars.
PKD probably begins in utero in most patients with the kidneys increasing in size and ultimately showing signs of disease in the fourth or fifth decade of life. At present there are methods of detecting PKD in utero. Approximately 25% of patients do not have a family history consistent with ADPKD, suggesting either that the genes responsible have a high mutation rate or that other environmental factors are at work.
Even though the specific gene defect responsible for PKD is unknown, the most common locus found by linkage studies is found on human chromosome 16p. Both in vivo and in vitro studies of ADPKD kidneys have suggested that a variety of disease manifestations (including accelerated renal epithelial cell growth, basement membrane abnormalities and mislocalized membrane proteins) are potentially important in the etiology and maintenance of renal cysts. Additional cysts involving the liver or spleen, and brain aneurysms, have been found to be present in about ten percent of ADPKD patients suggesting that the disorder affects organs other than the kidneys.
To discover the molecular basis for cyst formation relating to PKD, researchers have employed both in vitro and animal models. Previous in vivo and organ culture studies have implicated both accelerated renal epithelial cell growth and electrolyte transport abnormalities in the genesis of these cysts.
Monolayer cultures of both normal and polycystic human kidneys have similar limited life spans and can be passaged using either serum free or supplemented media. Virtually no cysts or hemicysts have ever been observed in these cultures. In contrast, MDCK and LLCPK are two immortalized normal renal cell lines that spontaneously form hemicysts and cysts in culture. Cyst formation has also been reported in cultures of normal human thyroid, mammary and lung cells. Normal and polycystic kidney epithelial cells, embedded in collagen can be induced to form cysts when stimulated with serum, forskolin, or epidermal growth factor. However, conditions enabling the differential expression of normal and polycystic kidney phenotypes using disbursed cells in culture has not been achieved.
In vivo experiments have been performed on a mouse strain (CPK) which is homozygous for a gene that causes a murine polycystic kidney disease. This murine model arose as a spontaneous mutation of the C57BL/6J strain housed at the Jackson laboratories, and newborn pups rapidly develop a severe form of polycystic kidney disease. Numerous proximal cysts are found in the kidney cortex of these CPK mice at birth.
These proximal cysts progressively enlarge and by day 10 the collecting duct segments of thousands of kidney tubules begin to dilate and develop into large fluid filled cysts throughout the kidney medulla. Impairments of renal functions parallel the enlargement of these collecting duct cysts. Affected mice become visibly lethargic due to azotemia around day 20-25 after birth and usually die of uremia and kidney failure before the 28th day.
Mechanistically, three types of abnormalities in normal kidney tubules have been implicated in the genesis and progression of cysts in PKD: (1) renal tubules begin to enlarge, forming a cyst, (2) additional cells line the nephron wall, and (3) a net change in renal fluid handling occurs allowing fluid to accumulate inside the cyst cavity.
Each of these three steps can be coded for by the same or different polycystic kidney disease genes. However, it is still unknown whether these observed phenomena are the direct result or secondary manifestation of altered genes in PKD. It is known that cysts arise from the progressive focal dilation of preexisting kidney tubules. Unfortunately, except for dialysis and transplantation, which are palliative, no curative treatment exists for PKD. A curative treatment for cystic disease, especially PKD, would therefore present an important medical breakthrough.